Ln 428

The human lung cancer cell line H1299 and the human glioblastoma cell line LN428 were obtained from the American Type Culture Collection (Manassas, VA, USA) and Trevigen (Gaithersburg, MD, USA), respectively. The 16HBE14o- cell line (Simian virus 40-transformed human bronchial epithelial cells) [22 (link)] was a gift from Dr. D.C. Gruenert (California Pacific Medical Center Research Institute, San Francisco, CA, USA) via Dr. T. Kaneko (Department of Internal Medicine, Yokohama City University, School of Medicine, Yokohama, Japan). The cells were maintained at 37°C in RPMI 1640 medium supplemented with 10% fetal bovine serum and penicillin/streptomycin under a 5% CO₂ atmosphere.

Eight human malignant glioma cell lines (LN-18, LN-229, LN-308, LN-428, U87MG, U251MG, U373MG, and D247MG) were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). All cell lines were cultured in Dulbecco's modified essential medium (DMEM, Invitrogen Corp., Grand Island, NY, USA) and supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen Corp., Grand Island, NY, USA), 2 mM glutamine, and gentamicin at 37°C in a humidified incubator with 5% CO₂. Experiments were performed in triplicate.

All glioblastoma cell lines [LN-229 (RRID:CVCL_0393), LN-308 (RRID:CVCL_0394), U-87MG ATCC (RRID:CVCL_0022), LN-428 (RRID:CVCL_3959), and T98G (RRID:CVCL_0556)] were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured under standard conditions. Glioblastoma-initiating cells (T325, T269, and S24) were established from freshly dissected glioblastoma tissue from adult patients after informed consent and cultured in neurosphere medium (DMEM/F12 medium, Life Technologies, Carlsbad, CA, USA) supplemented with B27 supplement, heparin (5 μg/ml), basic fibroblast growth factor (20 ng/ml), and epidermal growth factor (20 ng/ml) (22 (link)). All cell lines tested have an isocitrate dehydrogenase wild-type status. The SNP profiles matched known profiles or were unique. The cell lines were authenticated using Multiplex Cell Authentication by Multiplexion (Heidelberg, Germany) as previously described (23 (link)). Absence of mycoplasma infection is screened for on a regular basis.

The human glioma cell lines LN-18, LN-229, LN-319, T98G, U251, U138MG and LN-428 were purchased from the American Type Culture Collection (Manassas, VA, USA). The human glioma cell line LN-308 was provided by N. de Tribolet. Regular checks for authentity and freedom from infection, e.g. mycoplasms, were done according to the institutional guidelines at the German Cancer Research Center. Details concerning cloning, quantitative reverse transcription PCR analyses (including Table S3), sequencing (including Table S4), shRNA constructs for VEGFR-2, siRNA constructs for Raptor or Rictor and overexpression constructs for VEGFR-2 or PTEN including the respective controls are given in the Supplementary Information.